title: Insights into the mechanism of pore opening of acid-sensing ion channel 1a.
abstract: Acid-sensing ion channels (ASICs) are trimeric cation channels that undergo activation and desensitization in response to extracellular acidification. The underlying mechanism coupling proton binding in the extracellular region to pore gating is unknown. Here we probed the reactivity toward methanethiosulfonate (MTS) reagents of channels with cysteine-substituted residues in the outer vestibule of the pore of ASIC1a. We found that positively-charged MTS reagents trigger pore opening of G428C. Scanning mutagenesis of residues in the region preceding the second transmembrane spanning domain indicated that the MTSET-modified side chain of Cys at position 428 interacts with Tyr-424. This interaction was confirmed by double-mutant cycle analysis. Strikingly, Y424C-G428C monomers were associated by intersubunit disulfide bonds and were insensitive to MTSET. Despite the spatial constraints introduced by these intersubunit disulfide bonds in the outer vestibule of the pore, Y424C-G428C transitions between the resting, open, and desensitized states in response to extracellular acidification. This finding suggests that the opening of the ion conductive pathway involves coordinated rotation of the second transmembrane-spanning domains.
authors.affiliation: Renal-Electrolyte Division, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
authors.forename: Lindsey A
authors.initials: LA
authors.lastname: Tolino
authors.forename: Sora
authors.initials: S
authors.lastname: Okumura
authors.forename: Ossama B
authors.initials: OB
authors.lastname: Kashlan
authors.forename: Marcelo D
authors.initials: MD
authors.lastname: Carattino
mesh_terms: D062565:Acid Sensing Ion Channels; D019943:Amino Acid Substitution; D000818:Animals; D004220:Disulfides; D007202:Indicators and Reagents / Q000494:pharmacology; D017136:Ion Transport / Q000187:drug effects / Q000502:physiology; D008698:Mesylates / Q000494:pharmacology; D051379:Mice; D016296:Mutagenesis; D020125:Mutation, Missense; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D015222:Sodium Channels / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: C568267:ASIC1 protein, mouse; D062565:Acid Sensing Ion Channels; D004220:Disulfides; D007202:Indicators and Reagents; D008698:Mesylates; D009419:Nerve Tissue Proteins; D015222:Sodium Channels; C074502:methanethiosulfonate